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Expression and Purification of vMIP-II-TfN in Pichia pastoris |
WANG Feng, SUN Han-xiao, LI Xiu-ying, ZHANG Guang, MO Xue-mei |
Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China |
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Abstract Objective: To construct the pPIC-vMIP-II-TfN yeast expression vector and to express vMIP-II-TfN fusion protein. Method: PCR was employed to amplify the N-terminal half-molecular of human transferrin gene. Molecular cloning methods including digestion, ligation and transformation were used to construct the pPIC-vMIP-II-TfN yeast expression vector. The linearized pPIC-vMIP-II-TfN was transformed into Pichia pastoris X33 strain by electroporation. The transformant was induced by methanol to express target protein and ammonium sulfate precipitation, dialysis, and Ni-NTA affinity chromatography were used to purify the target protein. SDS-PAGE and Western blot was used to detect the expression and purification of vMIP-II-TfN. The activity of vMIP-II-TfN was detected by chemotaxis inhibition activity assay. Results: A 1.1kb IgG3-TfN DNA fragment containing the Xba I site was amplified and it was inserted into Xba I sites of pPIC-vMIP-II. After PCR identification and sequencing, the recombinant plasmid pPIC-vMIP-II-TfN was obtained successfully and the open reading frame was correctly. The linearized pPIC-vMIP-II-TfN by Sac I was transformed into Pichia pastoris X33 strain. After inducing with methanol for 72 h, the transformant was confirmed to express a recombinant protein with molecular mass of 48 kDa by SDS-PAGE and Western blot. After characterization, the vMIP-II-TfN fusion protein was successfully purified from the supernatant of the broth using ammonium sulfate precipitation, dialysis and affinity chromatography. Furthermore, in vitro bioacitivity assay indicated that vMIP-II-TfN has chemotaxis inhibition activity. Conclusion: The pPIC-vMIP-II-TfN yeast expression vector was constructed successfully. The transformant can express the vMIP-II-TfN fusion protein which had chemotaxis inhibition activity.
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Received: 29 June 2011
Published: 25 October 2011
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